Fig 2: Endocytosis inhibits the vesicular secretion of CD63, while CD63 expression induces the vesicular secretion of Lamp1, Lamp2, and AP-2 mu2.(A) Immunoblots of cell lysates and exosome-containing fractions (100K pellet) collected from uninduced FtetZ/CD63−/− cells carrying WT CD63, CD63-YQRF, CD9-YQTI, or CD63-AEMV transgenes. MW size markers in kDa. (B) Bar graph showing that strengthening the CD63 endocytosis signal inhibited its vesicular secretion, while mutationally inactivating the CD63 endocytosis signal increased its vesicular secretion. E/C ratios were normalized to 1 for the WT CD63 sample in this experiment. **P < 0.01. (C) Immunoblots of cell lysates and exosome-containing fractions (100K pellet) collected from dox-induced FtetZ/CD63−/− cells carrying WT CD63, CD63-YQRF, CD9-YQTI, or CD63-AEMV transgenes. MW size markers in kDa. (D) Bar graph showing that high-level expression led to all CD63 proteins displaying the same vesicular secretion (E/C ratio). E/C ratios were normalized to 1 for the WT CD63 sample in this experiment. denotes P value of >0.05. (E to G) Bar graphs of E/C ratios for (E) Lamp1, (F) Lamp2, and (G) mu2 in CD63 knockout cells carrying the WT CD63, CD63-YQRF, or CD9-YQTI transgenes, grown in the (blue) absence or (red) presence of dox. E/C ratios were in each case normalized to 1 for the -dox sample. ***P < 0.001, ****P < 0.0001. (H) Transmission electron micrographs of FtetZ/CD63−/− cells expressing a high level of CD63. Arrowheads point to the presumed neck of possible budding intermediates. Scale bar, 100 nm. (I) Flow cytometry histograms showing the surface-stained CD63 fluorescence of 293F cells grown overnight in the (blue) absence of bafilomycin or (red) presence of bafilomycin. (J) Bar graph showing the relative levels of cell surface CD63 staining (MFI) of 293F cells grown in the (blue) absence of bafilomycin or (red) presence of bafilomycin. **P < 0.01.

Fig 3: C. albicans promotes EGFR degradation through Ece1p and Als3p, predominantly mediated by the lysosomal pathway, and induces the recruitment of adaptor proteins Grb2, AP2M1, and HRS. (A–C) TR146 cells were infected with different C. albicans strains (wild-type [WT] SC5314, ece1Δ/Δ and als3Δ/Δ) or stimulated with PBS as a vehicle control for 2 h (MOI 10), 8 h (MOI 1), and 12 h (MOI 1). Cell lysates were electrophoresed on gradient gels to detect total EGFR and α-actin via Western blot. Data are representative of three independent experiments. (D and E) TR146 cells were pre-treated for 1 h with 0.25 µM of lysosomal inhibitor Bafilomycin A1 (BafA1) and 10 µM of proteasomal inhibitor MG-132 or DMSO as a vehicle control, and then treated with 100 ng/mL EGF as a positive control for 1 h or infected with SC5314 (MOI 5) for 10 h. Cell lysates were electrophoresed on gradient gels to detect total EGFR and α-actin via Western blot. Data are representative of three independent experiments. Dashed vertical lines indicate omitted, extraneous portions of blot images. (F and G) Relative protein levels in panels D and E were quantified using densitometry. Total EGFR was normalized to α-actin and expressed relative to the control (DMSO + water or DMSO + PBS), which was set to 100. Data are presented as mean values with standard error of the mean error bars. Statistical significance was determined using a parametric test (ordinary one-way ANOVA with Dunnett’s multiple comparison test). *P < 0.05. ns, not significant.

Fig 4: C. albicans induces EGFR phosphorylation at Y1045 and EGFR ubiquitination in TR146 oral epithelial cells in an ECE1-driven manner. (A) Schematic of EGFR phosphorylation and ubiquitination. Created with BioRender.com. (B) TR146 cells were infected with wild-type (WT) C. albicans (BWP17), an ECE1 null strain (ece1Δ/Δ), an ECE1 re-integrant strain (ece1Δ/Δ + ECE1), or a candidalysin null strain (ece1Δ/Δ + ECE1Δ184–279) for 4 h at an MOI of 10 or stimulated with PBS as a vehicle control. Lysates (10 µg) were electrophoresed on gradient gels to detect pEGFR Y1045 and α-actin via Western blot. Data are representative of three independent experiments. (C) TR146 cells were infected with WT C. albicans (SC5314), ece1Δ/Δ, ece1Δ/Δ + ECE1, ece1Δ/Δ+ECE1Δ184–279, and als3Δ/Δ for 6 h at an MOI of 5 or stimulated with PBS as a vehicle control. EGF 100 ng/mL was used as a positive control. Lysates were collected and EGFR was immunoprecipitated. Immunoprecipitation (IP) samples were electrophoresed on gradient gels to detect total EGFR and ubiquitin via Western blot. Data are representative of three independent experiments. Dashed vertical lines indicate omitted, extraneous portions of blot images. (D) Relative protein levels in panel C were quantified using densitometry. Ubiquitin was normalized to total EGFR and expressed relative to the control (PBS), which was set to 1. Data represent the average of three to seven independent experiments and are presented as mean values with standard error of the mean (SEM) error bars. Statistical significance was determined using the Kruskal–Wallis test with Dunn’s multiple comparison test. P < 0.05, ***P < 0.001. (E) TR146 cells were infected with WT C. albicans (SC5314), ece1Δ/Δ, and als3Δ/Δ for 6 h at an MOI of 5 or stimulated with PBS as a vehicle control. Lysates were collected and EGFR was immunoprecipitated. IP samples were electrophoresed on gradient gels to detect total EGFR, Grb2, AP2M1, and HRS via Western blot. Dashed vertical lines indicate omitted, extraneous portions of blot images. Data are representative of three independent experiments. (F–H) Relative protein levels in panel E were quantified using densitometry. Grb2, AP2M1, and HRS were normalized to total EGFR and expressed relative to the control (PBS), which was set to 1. Data represent the average of three to four independent experiments and are presented as mean values with SEM error bars. Statistical significance was determined using a parametric test (ordinary one-way ANOVA with Dunnett’s multiple comparison test). *P < 0.05. ns, not significant.